Cholesterol assay and reagents therefor

ABSTRACT

A cholesterol assay, using formamide, which achieves the simplicity of a direct test of a specimen sample, yet which also achieves a comparable specificity and accuracy of cholesterol assays involving multiple-extraction steps. The formamide destroys the cholesterol chromophore of a blank measurement, and this result may be compared with a companion cholesterol assay to eliminate the error caused by the chromophore of non-cholesterol chromogenic substances of the specimen; and the formamide also keeps the alcohol, which is used to prevent turbidity, from blocking the formation of the bilirubin portion of the noncholesterol chromogenic substances.

Elite States atent Denney Feb. 6, 1973 [75] Inventor: Jerry W. Denney,Carmel, Ind.

[73] Assignee: American Monitor Corporation, In-

dianapolis, lnd.

[22] Filed: April 30, 1971 [21] Appl. No.: 139,266

[52] US. Cl. ..23/230 B [51] 1nt.Cl. ..G0ln 33/16 [58] Field of Search..23/23O B; 252/408 [56] References Cited UNITED STATES PATENTS3,558,516 1/1971 Wybenga ..23/230 B X 3,615,232 10/1971 Parekh ct a1...23/230 B Primary Examiner-Morris O. Wolk Assislan l Examiner-R. M.Reese Atl0rneyRobert- A. Spray [57] ABSTRACT A cholesterol assay, usingformamide, which achieves the simplicity of a direct test of a specimensample, yet which also achieves a comparable specificity and accuracy ofcholesterol assays involving multiple-extraction steps. The formamidedestroys the cholesterol chromophore of a blank measurement, and thisresult may be compared with a companion cholesterol assay to eliminatethe error caused by the chromophore of non-cholesterol chromogenicsubstances of the specimen; and the formamide also keeps the alcohol,which is used to prevent turbidity, from blocking the formation of thebilirubin portion of the noncholesterol chromogenic substances.

11 Claims, No Drawings CHOLESTEROL ASSAY AND REAGENTS THEREFOR and OtherFactors as to Cholesterol Present in Blood Serum, and as to the Need forAccurate Measurement Thereof.

Cholesterol, which is one of the fatty materials found in the bloodserum, and which is formed primarily in the liver, is generallyacknowledged to be very significant as a factor in various diseaseprocesses of the human body, including diseases of the heart, vascularsystem, etc.

The implication of cholesterol in the etiology and prognosis ofatherosclerosis and heart disease has stimulated an enormous growingliterature on the subject, much of it asserting and acknowledging thatthere appears to be a statistically significant relation between highserum cholesterol levels and the incidence of coronary artery disease;and this relation has made it considered to be desirable to maintainlow, or at least low normal, levels of cholesterol in serum.

Thus, accurate cholesterol measurement of serum cholesterol is verydesirable, since if an individual is falsely diagnosed to have an undulyhigh cholesterol level, he may be wrongfully placed on an unpleasant orexpensive corrective diet; or, if the diagnosis is too low, a person whomight need the benefit of such corrective diet may go untreated.

Correct cholesterol measurement is also needed to avoid a falsely highcholesterol diagnosis which might cause a thyroid disease to beunsuspected or overlooked, for certain thyroid diseasescharacteristically cause a low cholesterol level. Hyperthyroidism isassociated with hypocholesterolemia to such an extent that serumcholesterol measurement has been used to monitor thyroid status; and theneed for cholesterol measurement accuracy is thus again noted.

Diseases of the liver alter cholesterol levels, and the measurement ofserum cholesterol yields information relating to the functioning of theliver, such as the ability of the liver cells to manufacture variouscompounds.

Not only is the serum cholesterol level of an individual important butalso it is important that the accuracy of the assay be close enough soas to reliably so detect any change which might occur from day to day,week to week, or month to month; since it is important to follow drugand diet therapy, and to detect cholesterol changes induced thereby,and/or changes of body response. If the test performed is notsufficiently reliable and specific, variation in some interfer ingsubstance might be falsely interpreted as a trend in cholesterol level,particularly since the interfering substances themselves change inconcentration from time to time, and not at all always proportionatelyto any change of cholesterol.

Thus, although a sufficiently accurate measurement of cholesterol inbody fluids has been long recognized to be a valuable and vitallydesired diagnostic factor, its measurement has continued to be aproblem. The prior art has attempted various methods of cholesterolmeasurement; but an inherent and continuing problem has been that of themasking or interference of other substances in the specimen to beassayed. Thus direct or non-sequential methods have not avoided theuncertainty or error attributable to those other substances; andmulti-step or sequential methods for extracting the non-cholesterolsubstances have caused higher assay costs, and their complexitycontributes to an increased chance of error.

As a vividly illustrative criticism of the thorough dissatisfaction andrecognized unreliability of the cholesterol assays of the prior art, theYale professor who is Chairman of the Yale University School of Medicine(Section of Laboratory Medicine) has very recently written: I believethat more than per cent of cholesterol values currently issued tophysicians are unsuitable for the practice of medicine or clinicalresearch. (The New England Journal of Medicine,

p. 394, (Feb. 18, 1971).)

Perhaps the reason for this alleged unsuitability is the fact that thosemethods which are most reliable in the art prior to the presentinvention are also the most complicated and time consuming and thus arenot widely used. That these methods would be used in spite of theirunreliability problems might be explained by the fact that manystandardization and quality control programs do not include sources ofinterference in the standardization and control specimens; and oftenperformance is judged in comparison to other laboratories in theprograms who may also be doing inaccurate test- PRIOR ART METHODOLOGYFOR CHOLESTEROL ANALYSIS The number of procedures available for thedetermination of cholesterol and its esters is multitudinous, and yetthe fundamental chemistry on which they depend may be considered to bebasically similar or in many respects identical. It has been known for along time that the two reactive centers in the cholesterol molecule arerepresented by the double bond and the hydroxyl group; of these, thedouble bond is the most important in the formation of color orchromophore.

Cholesterol reacts with strongly acid reagents to produce coloredsubstances, chiefly cholestadiene sulfonic acids. In virtually allprocedures, acetic acid and acetic anhydride are used as solvents anddehydrating reagents, and sulfuric acid or sulfuric acid andptoluenesulfonic acids are used as dehydrating and ox idizing reagents.in some procedures, the reaction of COLOR REACTIONS 0F CHOLESTEROL acholesterol (2 molecules) H10 -l- BK 3,5-oholestadlene (2 molecules) -11bi- BK bis-3,6-eholvstadiene H1804 2504 H20 2H2Obio-cholestadienylbis-cholestadienylmonosulfonie acid dlsulfonlc acidGREEN COLORED RED COLORED CHROMOPHORE CHROMOPHORE Llebermann-Burchard"Salkowski" Tietz, N. W., Fundamentals of Clinical Chemistry, p. 353 (W.B. Saunders, Philadelphia, Pa. 1970) According to this scheme,cholesterol is first attacked by strongly acid reagents, generalized asHX, where X" would be, for example, the sulfate ion or the acetylradical. Such reagents first remove a molecule of water, then oxidizethe intermediate to produce 3,5- cholestadiene (two double bonds). Theoxidizing agent is usually sulfuric acid, which is converted to sulfurdioxide. The cholestadiene is attacked still further to form the dimer,bis-cholesta-3, S-diene, and is finally converted by the excess sulfuricacid, to a monoor disulfonic acid which is a highly colored molecule.Depending on the relative concentration of the oxidant, sulfuric acid,one gets either a green color (Liebermann-Burchard) due to amonosulfonic acid, or a red color (Salkowski) due to the formation ofdisulfonic acid. The addition of iron or other metal ions favors theformation of the red color due to cholestadiene disulfonic acid. AsVanzetti points out, the reactions are not entirely specific, nor canthey be controlled with sufficient precision to yield always the exactproducts shown, but with proper care, the reactions can be the basis ofan accurate reproducible analytical system. Insofar as the chemistry isconcerned, there is little reason to prefer a red color reaction over agreen color reaction, except that the red reactions generally produce acompound with a higher molar absorbance than the green." Choice of aspecific procedure is, therefore, based on other issues. These mayinclude the interference by such substances as bilirubin, byconsiderations of speed and facilities available, and factors such asthe manipulations required in the sample preparation.(4. lbid.)

SINGLE-STEP PROCEDURES In these methods, the sample is not purified toany degree, and the colorimetric reaction is performed directly on serumor plasma. These methods are liable to several errors. First, theever-present protein may be charred during color development, thusproducing a color which may be mistaken as coming from cholesterol.Secondly, such procedures may also suffer from interferences due tonon-specific color-forming substances or chromogens, includingbilirubin, hemoglobin, and protein, and by instability of the finalcolored product. It can be said as to these procedures that they arerapid, and that they require the least degree of manipulation, and thatfrom a procedural standpoint they are perhaps best suited to automation;however, because of the above problems they are of doubtful accuracy formore than screening purposes. Examples of this type of procedure are themethods of Pearson, Stern, and McGavick,-"(5. Pearson, .1., Stem, S.,and McGavack, T.l-l.: Anal. Chem. 251813, 1953.) and Zlatkis, Zak andBoyle. (6. Zlatkis, A., Zak, B., and Boyle, A. 1.: J. Lab. Clin. Med.,41 :486, 1953.)

TWO-STEP PROCEDURES These methods introduce an extraction step, whichextracts and partially isolates the cholesterol chromogen beforechromophore or color development. For this reason, some of theinterfering chromogens, especially protein, are removed, but the methodsstill are subject to error caused by non-specific chromogens, such asbilirubin which is extracted along with the cholesterol, and thus stillprovides a chromophore which is mistakenly measured as cholesterolchromophore. Of several methods in this class, that of Carr andDrekter"(7. Carr, 1.1., and Drekter, 1.1.: Clin Chem. 2:353, 1956.) isperhaps the best. Except for highly icteric samples (high bilirubinlevels), it gives results that are in close correspondence to thoseobtained by more precise three-step methods; however, icteric samplesare encountered with enough frequency as to create problems in routineanalysis.

THREE-STEP PROCEDURES These involve an extraction of the cholesterolfollowed by a saponification of the esters before color development.Consequently, they do not suffer from serious error because of protein.Furthermore, the saponification step tends to destroy nonspecificchromogens such as bilirubin, resulting in significantly increasedaccuracy over two-step methods. This class of procedures is bestexemplified by the method of Abell et al.;*- (8. Abell, L.L., Levy,B.B., Brodie, B.B., and Kendall, F.E., J.Biol.Chem., 195, 357, 1952.)Henry R.J.: Clinical Chemistry, Principles and Technics, New York,l-loeber Division, Harper and Row Publishers, 1964.) which is acceptedas a standard method in many laboratories, in spite of its complexity,as it is the least complicated method which is free of error fromextraneous chromogens, prior to the present invention.

FOUR-STEP PROCEDURES These are the most complicated, but the mostreliable procedures of the prior art, if their complexity does notintroduce errors-which that very complexity makes possible. Thecholesterol is extracted, the esters are saponified, and the totalcholesterol is then further purified by collection as the digitonide.The digitonide is decomposed by saponification, which again frees thecholesterol, and the product of this step is subject to colordevelopment.

By introduction of the digitonin step, the effect of nonspecificchromogens is considerably reduced or eliminated, but then thecholesterol digitonide must be decomposed and the digitonin removed;otherwise, it too will give a positive reaction with the usual reagentssince the digitonin structure includes a sterol ring as doescholesterol. Cholesterol and digitonin, however, do not have the samechromogenicity, and it is therefore advisable to remove the digitoninbefore performing the chromogenic reaction. Four-step procedures involvemany separate steps, each of which requires careful control;nevertheless, methods of the multi-step type may be of high accuracy,and either the method of Schoenheimer and Sperry' (l0. Schoenheimer, R.,and Sperry, W.M.: J.Biol.Chem, 106:745, 1934.) or of Sperry and Webb(11. Sperry, W.M., and Webb, M.: J.Biol.Chem., 187:97, 1950.) are themost widely accepted reference methods for the determination ofcholesterol.

(Many other methods in each of the above categories could be cited, mostof which represent slight modifications of those fundamentalprocedures.)

While multi-step measurements of cholesterol can give adequate resultswhen properly used, serious errors may be encountered due to incompleteextraction resulting in poor recovery of cholesterol and by volumetricerrors due to the number of volumetric measurements made. Moreover,multi-step measurements obviously have the disadvantages of beinglaborious and time-consuming, and therefore they tax the resources of abusy clinical laboratory to such an extent that direct methods arepresently in wide use, even in spite of their lack of specificity andeven in spite of their subjectivity to error from chromogenic substancesother than cholesterol which are present in serum. The most desirablemethod would be one which may be performed directly upon serum, butwhich agrees more closely with more complicated multi-step methods suchas that of Abell.

The present invention achieves a solution to goals which heretofore haveappeared to be in effect contradictory to one another; that is, as nowdiscussed, the present invention achieves substantially the simplicityof a direct test while nevertheless achieving specificity and accuracycomparable to those involving multipleextraction steps.

THE PRESENT INVENTION It has been discovered that when formamide in theproper concentration is added to a cholesterol reagent used to assay asample of serum, under the proper conditions, the chromophore formedfrom the cholesterol of the serum is destroyed while the bilirubin,hemoglobin, and protein chromogens of the specimen react much as they doin the cholesterol reagent alone. Thus according to the concepts of thepresent invention, there is performed a companion blankspectrophotometric or colorimetric in which the effect of thesenon-cholesterol substances is in effect subtracted from thecorresponding spectrophotometric or colorimetric test in which both thecholesterol and these substances react. By performing such a blank, theerror contributed by these non-cholesterol chromogenic substances issufficiently minimized in the assay, achieving satisfactory precision oraccuracy of the measurement of cholesterol itself, unmasked by thechromophore of the other chromogenic substances present yet withoutmultiple extraction steps.

(Other substances have been found which in some way prevent theformation of the color of chromophore from cholesterol chromogen.Alcohols, for instance, when used in the proper concentration have thateffect. However, alcohols when used alone also prevent the formation ofa chromophore from the bilirubin chromogen, and therefore when usingonly alcohol in a blank, bilirubin interference remains as acontributing error in the test; and a comparison of a blank measurementwith a test measurement would give a total of bilirubin and cholesterolinstead of specifically the desired measurement of cholesterol itself.)

Formamide when present in a cholesterol reagent destroys thechromophore-forming reaction of the cholesterol chromogen at roomtemperature; however, the bilirubin chromogen does not completely formits chromophore as required for proper blank correction, at roomtemperature. It has been found that the formamide-cholesterol reagentmust reach an elevated'temperature of about 80 C before sufficientchromophore formation from the bilirubin chromogen reaction will takeplace. Consequently, if formamide and the I cholesterol reagent weremixed in a constant temperature water bath commonly used in certainwidely used cholesterol methods, the bilirubin chromophore might notdevelop, and thus the contribution of the bilirubin chromogen may not beobserved and would not be subtracted. However, it has been found that ifthe formamide is added to the blank tube at room temperature withoutconstant-temperature incubation, the desired reaction occurs, that is,the green bilirubin chromophore forms while the cholesterol chromophoreis eliminated. In this case the formamide serves two functions: First,one effect of its reaction with the cholesterol reagent is that itraises the temperature of the mixture to the proper range for thereaction, thus eliminating the necessity of incubation in a hightemperature bath; and second, it has the desired effect on the bilirubinand cholesterol chromogen formation.

While formamide used alone permits the effect of chromophore formationfrom the bilirubin and other non-cholesterol chromogen and inhibitscholesterol chromophore development, it has been found that someturbidity develops in the blank in the presence of certain sera due tothe presence of serum and that this turbidity falsely elevates theblank, by increasing the absorbence of the blank during colorimetric orspectrophotometric measurement, thus causing the turbidity to bemistaken for interfering chromogens, unless the turbidity is somehowaccounted for or avoided.

This problem has been avoided in this invention by the concept ofincluding in the mixture a high molecular weight alcohol (an alcoholwith eight to 16 inclusive, carbon atoms per molecule). As discussedabove, when alcohol is used alone in the blank determination, it doesdestroy the cholesterol chromophore, but it has the undesired effect ofadditionally destroying the desired formation of the bilirubinchromophore; however, it has been found that when a high molecularweight alcohol is used in conjunction with formamide, the desiredbilirubin chromophore does form in the desired fashion and thatturbidity is prevented from forming.

Formamide and a high molecular weight alcohol thus serve the desiredfunction of preventing cholesterol chromophore development whileallowing bilirubin chromophore development in a way in which turbidityis not observed; however, since the high molecular weight alcohols donot mix with the formamide, the formamide and alcohol must be added intwo separate steps to the blank. This separateness of procedural stepsis avoided in the present invention by the addition of the highmolecular weight alcohol to the glacial acetic acid acetic acid sulfuricacid reagent. It has been found that a final concentration ofapproximately 2% V/V of the alcohol with respect to the cholesterolreagent is effective in eliminating the turbidity sometimes observed inthe formamide-containing blank .measurement and that inclusion of thealcohol in this reagent greatly simplifies the procedure over theprocedure using separate addition of the alcohol.

Various high molecular weight alcohols differ in their effectiveness ineliminating turbidity; generally the higher molecular weights are moreeffective and can thus be used in lower concentrations.

1ST EMBODIMENT Blank reagent: formamide Cholesterol reagent:

166 ml H,SO.;

500 ml glacial acetic acid, cool to room temperature;

add 500 ml acetic anhydride;

add 12 ml tridecyl alcohol.

The reagents are used in the following assay using a blank tube and atest tube:

Add 0.1 ml serum to both tubes. Add 1.0 ml Blank Reagent to the blanktube only. Add 2 ml of Cholesterol Reagent to the blank tube and 3 ml tothe test. The blank tube is allowed to stand for minutes at roomtemperature while the test is incubated at 37 C for l0 minutes afterwhich the absorbence of the test at 620 mu is measured against the blankwhich is set to zero absorbence.

The absorbence of the test so measured is proportional to theconcentration of cholesterol in serum, for the interfering chromogenscontribute equally to both test and blank; and since the blank is set atzero absorhence, the absorbence of the test is a measurement of thedifference between (or in effect a subtraction of) blank from test.

b 2ND EM BODIM ENT Same as lst Embodiment, except 20 ml of isononylalcohol is used instead of the tridecl alcohol.

3RD EMBODIMENT As in the lst Embodiment, except ml decyl alcohol is usedinstead of tridecyl alcohol.

b 4TH EMBODIMENT As in the lst Embodiment, except 10 ml hexadecylalcohol is used instead of tridecyl alcohol.

5TH EMBODIMENT Blank Reagent l: Octyl, .nonyl, decyl, tridecyl, orhexadecyl alcohol.

Blank Reagent ll: Formamide.

Cholesterol Reagent: As in lst Embodiment, except the alcohol isomitted.

The reagents are used in the following assay using a blank tube and atest tube;

Add 0.5 ml Blank Reagent I and 0.5 ml Blank Reagent II to the blank tubeonly. Proceed as in lst Embodiment beginning with the addition of theCholesterol Reagent.

CONCLUSION An assay according to the novel concepts of the inventionthus provides the multiple advantages of both simplicity correspondingto direct or one-step tests and accuracy of multiple-step ormultiple-extraction tests, and also avoiding certain error-contributingaspects of the multiple-step processes. Thus the often-incompatiblegoals of both speed and accuracy are provided, along with relative easeand convenience of the test procedures, elimination of time and error ofextraction steps while nevertheless attaining in effect the result ofhaving fully performed such extractions to avoid a masking or othereffect of the other chromophoreforming chromogens usually present in thesample or specimen being assayed.

Accordingly, it will thus be seen from the foregoing description of theinvention according to the embodiments of the invention herein setforth, that the present invention provides a new and useful assayyielding quantitative determination of cholesterol in serum, plasma, orother biological material to be tested, and provides a method andreagents therefor, all having desired advantages and characteristics,and accomplishing the objects of the invention, including the objectsand advantages hereinbefore pointed out and 'others which are inherentin the invention.

It will be understood that modifications and variations of the generaland specific concepts of the overall assay may be effected withoutdeparting from the novel concepts of this invention; accordingly, theinvention is not to be considered limited to the specific form orembodiments set forth herein for the purpose of disclosing andillustrating the inventive concepts discovered and herein applied.

What is claimed is:

l. A process for the quantitative colorimetric or spectrophotometricdetermination of cholesterol in serum, plasma, or other biologicalmaterial to be tested, wherein the improvement comprises addingformamide to said material for preventing the formation of a chromophorefrom the cholesterol chromogen in the sample being assayed.

2. The process as set forth in claim 1 in which the forrnamide is addedto a first portion of said material to prevent the said formation of thecholesterol chromophore to provide a blank measurement, and in whichforrnamide is omitted from a second portion of said material to providea test measurement, and comparing the blank measurement with the testmeasurement, whereby a quantitative determination of the cholesterolitself is achieved.

3. A process for the quantitative determination of cholesterol in serum,plasma, or other biological material to be tested, wherein theimprovement com- 0 prises adding formamide and a high molecular weightalcohol to said material for preventing the formation of chromophorefrom the cholesterol chromogen in the sample being assayed, whilepermitting the formation of the chromophore from the bilirubinchromogen.

4. The process as set forth in claim 3, the alcohol having from eight tosixteen carbon atoms per molecule.

5. The process as set forth in claim 3 in which the alcohol is tridecylalcohol.

6. The process as ser forth in claim 3 in which the alcohol is hexadecylalcohol.

7. The process as set forth in claim 3 in which the high molecularweight alcohol is added to the cholesterol reagent.

8. A process for the quantitative determination of cholesterol in serum,plasma, or other biological material to be tested, wherein theimprovement comprises adding formamide to block the formation of thechromophore from the cholesterol chromogen in a first portion of thesample while causing the non-cholesterol chromogenic substances in thesaid first portion of the sample to produce their chromophores,

and in a second portion of the sample the step of causing the formationof chromophore from the samples substance including both its saidnoncholesterol chromogenic substances and its cholesterol chromogen, byomitting forrnamide,

and comparing the chromophore of the two sample portions toquantitatively determine the cholesterol content of the sample by thecontrast of the chromophore produced in the two sample portions.

9. A process according to claim 8, in which a mixture of acetic acid,acetic anhydride, and sulfuric acid is added to the first and secondportions to cause the noncholesterol chromogenic substances in the saidfirst I UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.5,188 Dated February 6, 1973 Inventor(s) s y Denney It is certified thaterror appears in the above-identified patent I and that said LettersPatent are hereby corrected as shown below:

Column 1, lines 3 through 6, should appear in bold type.

"tridecl" should read tridecyl This certificate supersedes Certificateof Correction d -October 23, 1973.

Signed and sealed this 9th day of July 1974;

(SEAL) Attest:

McCOYYM. GIBSON,- JR. I ,C. MARSHALL DANN Attesting Officer 4 oCommissioner of Patents Column 5, line 32, after "colorimetric" insertmeasurement Column 7, line 24, cancel "b"; line 32, cancel "b". Column8, line 66, "substance" should read substances last line, before"formamide" insert the Column 7, line 27,

FORM PO-1050 (10-69) US COMM-DC 603764 69 ".5. GOVERNMENT PRINTINGOFFICE I9, 0-366-334,

1. A process for the quantitative colorimetric or spectrophotometricdetermination of cholesterol in serum, plasma, or other biologicalmaterial to be tested, wherein the improvement comprises addingformamide to said material for preventing the formation of a chromophorefrom the cholesterol chromogen in the sample being assayed.
 2. Theprocess as set forth in claim 1 in which the formamide is added to afirst portion of said material to prevent the said formation of thecholesterol chromophore to provide a blank measurement, and in whichformamide is omitted from a second portion of said material to provide atest measurement, and comparing the blank measurement with the testmeasurement, whereby a quantitative determination of the cholesterolitself is achieved.
 3. A process for the quantitative determination ofcholesterol in serum, plasma, or other biological material to be tested,wherein the improvement comprises adding formamide and a high molecularweight alcohol to said material for preventing the formation ofchromophore from the cholesterol chromogen in the sample being assayed,while permitting the formation of the chromophore from the bilirubinchromogen.
 4. The process as set forth in claim 3, the alcohol havingfrom eight to sixteen carbon atoms per molecule.
 5. The process as setforth in claim 3 in which the alcohol is tridecyl alcohol.
 6. Theprocess as ser forth in claim 3 in which the alcohol is hexadecylalcohol.
 7. The process as set forth in claim 3 in which the highmolecular weight alcohol is added to the cholesterol reagent.
 8. Aprocess for the quantitative determination of cholesterol in serum,plasma, or other biological material to be tested, wherein theimprovement comprises adding formamide to block the formation of thechromophore from the cholesterol chromogen in a first portion of thesample while causing the non-cholesterol chromogenic substances in thesaid first portion of the sample to produce their chromophores, and in asecond portion of the sample the step of causing the formation ofchromophore from the sample''s substance including both its saidnon-cholesterol chromogenic substances and its cholesterol chromogen, byomitting formamide, and comparing the chromophore of the two sampleportions to quantitatively determine the cholesterol content of thesample by the contrast of the chromophore produced in the two sampleportions.
 9. A process according to claim 8, in which a mixture ofacetic acid, acetic anhydride, and sulfuric acid is added to the firstand second portions to cause the non-cholesterol chromogenic substancesin the said first portion of the sample to produce their chromophoresand also to cause the formation of chromophore from the sample in thesecond portion of the reaction.
 10. The process according to claim 9 inwhich the mixture includes a high molecular weight alcohol.